Post-translational modification (PTM) is a fundamental mechanism for modulating protein function. One such PTM with increasingly recognized significance is protein lysine (Lys) acetylation. Like Tyrosine (Tyr)/Serine (Ser)/Threonine (Thr) phosphorylation, Lys acetylation is a reversible biochemical process, where a lysine acetyltransferase adds an acetyl group onto the ε-amine of a lysine residue. A deacetylase acts in the opposite way to remove it. Initially discovered in histones, lysine acetylation has also been observed recently in a very large number of other proteins, suggesting its diverse regulatory functions in the cell. There is mounting evidence that aberrant lysine acetylation is implicated in many disease conditions such as cancer and neurological disorders. Therefore, the study of lysine acetylation biology is of great importance and will lead to continuous therapeutic innovations.
Although many years of intensive research have firmly established a broad role for lysine acetylation as a histone epigenetic mark affecting chromatin structure and function, the effects of most individual acetylation events—especially those identified more recently—remain to be elucidated. Recent research in genetics, cell biology and especially proteomics has identified lysine acetylation in a large number of non-histone proteins. The functions of most of these modifications are unknown. As a major limiting factor, the study of protein lysine acetylation is often hindered by a lack of homogeneous protein samples containing the acetylated lysine (“Lys-Ac”) residue(s) of interest. Such homogenously acetylated proteins would be invaluable reagents for discerning the structural and functional effects of a particular Lys-Ac PTM via biophysical and biochemical means.
Several methods are useful to prepare site-specific modified proteins, such as unnatural amino-acid mutagenesis using the amber stop codon/suppressor tRNA pair, and protein chemical synthesis, but there remain significant technical barriers for the wide use of these methods by the bioscience research community at large. For example, unnatural amino acid mutagenesis is not widely available because it is a proprietary technology, and protein chemical synthesis requires extensive expertise of a well-trained chemist and is technically very challenging as well as labor-intensive.
Another known method is based on the combined use of unnatural amino acid mutagenesis and chemical modification to generate an acetyl-lysine analog but the optical purity is lost on the modified amino acid. Enzymatic Lys acetylation of recombinant proteins would appear to be an attractive approach. However, given the promiscuity of lysine acetyltransferases and the incomplete nature of enzymatic reactions, it is difficult if not impossible to isolate or characterize the desired acetylation product for structural and functional investigations.
As for other enzymatic reactions that do offer the necessary specificity, they often must work in the context of large macromolecular complexes such as with the help of other adaptor molecules, which renders them of little practical value.
A chemical approach for selective installation of acetylated Lys residues in recombinant proteins is therefore highly desired. Evidently, with the presence of many possible lysine residues in a normal protein, direct acetylation of a particular lysine residue is chemically not feasible.
The unique reactivity of the thiol group of cysteine (Cys) as a soft nucleophile has been exploited extensively for selective protein modification. Previously, a chemical method was developed to install a close isosteric analog of N-methyl-lysine into recombinant proteins (See Scheme I below). The method is based on a traditional alkylation reaction of the thiol group of a Cys residue with aminoethyl bromide or chloride, yielding aminoethylcysteine which is known to be a lysine equivalent.
When the alkylating agent is changed to N-methylaminoethyl halide, an N-methylated aminoethylcysteine residue or N-methyl thiaLys (“thiaLys(Me)”) is generated, which has been shown to be functionally similar to the natural Lys(Me).

As such, similar strategies have been proposed to prepare a close mimic of the native Lys(Ac) residue by making use of the unique reactivity of the cysteine thiol group. However, attempts to alkylate the Cys thiol with a similar N-acetyl-aminoethyl bromide and N-acetyl-aziridine failed to give the desired product (See Scheme IIa).
Further efforts in the prior art led to the development of methylthiocarbonyl-aziridine as the alkylating agent to afford methylthiocarbonyl-thiaLys as an N-acetyl-Lys mimic (See Scheme IIb). Although the alkylation reaction was successful and the resultant methylthiocarbonyl-thiaLys was shown to be recognized by a bromodomain-containing protein and by anti-acetyl-Lys antibodies, the presence of a large sulfur atom between the carbonyl and methyl in the thiocarbamate moiety makes it only a distant analog of the acetamide part in Lys(Ac). From both steric and electrochemical viewpoints, there are obvious and considerable differences between the acetyl and methylthiocarbonyl group (see structure in Scheme IIb), which may explain why the thiocarbamate modification is resistant to histone deacetylase cleavage.

Clearly, analogous to 2-aminoethyl-cysteine (i.e., thialysine) and N-methyl-thialysine being ideal mimics of lysine and N-methyl-lysine respectively, the best mimic of Lys(Ac) would still be N-acetyl-4-thialysine or sLys(Ac) in which the thioether linkage is a close isosteric replacement of the γ-methylene group in natural Lys(Ac). As the position of this substitution is rather far away—by 2 carbon atoms—from the acetamide nitrogen, little differences are expected between this Lys(Ac) mimic and its natural counterpart in their exhibited physicochemical and biochemical properties. Unfortunately, current attempts to use a similar alkylation reaction with N-acetyl-aminoethyl bromide or iodide and N-acetyl-aziridine are unsuccessful at producing acetyl-thialysine with acceptable yields and selectivity.
Furthermore, nucleophilic substitution with an alkyl halide or the equivalent aziridine compound (Scheme IIa) cannot be used to selectively alkylate the thiol under conditions that are acceptable for a protein, so a different alkylation method must be provided.
Accordingly, there is a need to provide a method for alkylating the thiol group of Cys to obtain a Lys(Ac) mimic that overcomes or ameliorates one or more disadvantages disclosed above. Additionally, such a method will also be useful for installing other modifications (e.g., pegylation and ubiquitination) onto a thiol-containing compound such as a peptide or a protein.